Lactam Compound

ABSTRACT

The present invention provides the compound (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)- 1 -amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, compositions thereof, and methods for using the same.

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceutical and organicchemistry and is concerned with a compound which inhibits β-amyloidpeptide release and/or its synthesis.

BACKGROUND OF THE INVENTION

Certain lactams, which inhibit β-amyloid peptide release and/or itssynthesis, and accordingly, are useful for treating Alzheimer's disease,are described in PCT Application No. PCT/US97/22986.

The compound of the present invention(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneis useful for inhibiting β-amyloid peptide release and/or its synthesis,and, accordingly, is useful in treating Alzheimer's disease and hasadvantageous efficacy and safety properties.

SUMMARY OF THE INVENTION

This invention provides the compound(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.

In one of its method aspects, this invention is directed to a method forinhibiting β-amyloid peptide release and/or its synthesis comprisingadministering to a patient in need thereof with an effective amount of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.In a particular method embodiment, the present invention provides amethod for treating Alzheimer's disease comprising administering to apatient in need thereof with an effective amount of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.The present invention also provides a method for preventing orinhibiting the progression of Alzheimer's disease comprisingadministering to a patient in need thereof with an effective amount of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneand a pharmaceutically acceptable diluent. Such compositions are usefulfor inhibiting β-amyloid peptide release and/or its synthesis, includingthe treatment of Alzheimer' disease.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms below have the meanings indicated:

The term “ee” or “enantiomeric excess” refers to the percent by whichone enantiomer, E₁, is in excess in a mixture of both enantiomers(E₁+E₂), as calculated by the equation ((E₁−E₂)÷(E₁+E₂))×100%=ee. As iswell known in the art, enatiomeric excess can be determined by capillaryelectrophoresis and by chiral HPLC of the compounds or derivativesthereof.

Herein, the Cahn-Prelog-Ingold designations of (R)- and (S)- and thedesignations of L- and D- for stereochemistry relative to the isomers ofglyceraldehyde are used to refer to specific isomers.

The compound of the present invention can be prepared as describedbelow. In the Schemes below, all substituents, unless otherwiseindicated, are as previously defined and all reagents are well known andappreciated in the art.

In Scheme 1, step 1, N-methylphenethylamine of formula (1) is acylatedwith a suitable bisalkoxycarbonylacetate transfer reagent to give acompound of formula (2). N-methylphenethylamine is commerciallyavailable and is readily prepared by the reaction of a 2-bromo or2-chloroethylbenzene, under conditions well known and appreciated in theart, with an methylamine. A suitable bisalkoxycarbonylacetate transferreagent is one in which R₄ is C₁-C₄ alkyl and transfers abisalkoxycarbonylacetyl group to the compound of formula (1), such as,bisalkoxycarbonylacetic acids and bisalkoxycarbonylacetyl chlorides.(See Ben-Ishai, Tetrahedron, 43, 439-450 (1987)).

For example, the compound of formula (1) is contacted with a suitablebisalkoxycarbonylacetic acid to give a compound of formula (2). Suchcoupling reactions are common in peptide synthesis and synthetic methodsused therein can be employed. For example, well known coupling reagentssuch as carbodiimides with or without the use of well known additivessuch as N-hydroxysuccinimide, 1-hydroxybenzotriazole, etc. can be usedto facilitate this acylation. Such coupling reactions often use asuitable base to scavenge the acid generated during the reaction.Suitable bases include, by way of example, triethylamine,N,N-diisopropylethylamine, N-methylmorpholine and the like. The reactionis conventionally conducted in an inert aprotic polar diluent such asdimethylformamide, methylene chloride, chloroform, acetonitrile,tetrahydrofuran and the like. Typically the reaction is carried out attemperatures of from about O₂C to about 60° C. and typically requirefrom about 1 to about 24 hours. Upon reaction completion, the product offormula (2) is recovered by conventional methods including extraction,precipitation, chromatography, filtration, trituration, crystallizationand the like.

Alternatively, for example, the compound of formula (1) is contactedwith a suitable bisalkoxycarbonylacetyl chloride to give a compound offormula (2). Such acid chlorides are readily prepared from thecorresponding acids by methods well known in the art, such as by theaction of phosphorous trichloride, phosphorous oxychloride, phosphorouspentachloride, thionyl chloride, or oxalyl chloride, with or without asmall amount of dimethylformamide, in an inert solvent such as, toluene,methylene chloride, or chloroform; at temperatures of from about 0-80°C. The reaction is typically carried out for a period of time rangingfrom 1 hour to 24 hours. The acid chloride can be isolated and purifiedor can often be used directly, that is, with or without isolation and/orpurification. Such acylation reactions generally use a suitable base toscavenge the acid generated during the reaction. Suitable bases include,by way of example, pyridine, triethylamine, N,N-diisopropylethylamine,N-methylmorpholine and the like. The reaction is conventionallyconducted in an inert aprotic polar diluent such as methylene chloride,chloroform, tetrahydrofuran and the like. Typically the reaction iscarried out at temperatures of from about −20° C. to about 80° C. andtypically require from about 1 to about 24 hours. Upon reactioncompletion, the product of formula (2) is recovered by conventionalmethods including extraction, precipitation, chromatography, filtration,trituration, crystallization and the like.

In Scheme 1, step 2, a compound of formula (2) is cyclized to give acompound of formula (3).

For example, a compound of formula (2) is contacted with a acid, such asmethanesulfonic acid or sulfuric acid. The reaction is typically carriedout using the selected acid as a solvent. Typically the reactants areinitially mixed at temperatures of from about −20-C to about O₂C andthen allowed to warm to temperatures of about ambient temperature toabout 60-C. The cyclization reaction typically require from about 12 toabout 72 hours. Upon reaction completion, the product of formula (2) isrecovered by conventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization and the like.

In Scheme 1, step 3, a compound of formula (3) is deprotected to give acompound of formula (4).

The removal of such alkoxycarbonyl amine protecting groups is well knownand appreciated in the art. For example see, Protecting Groups inOrganic Synthesis, Theodora Greene (1^(st) and 2^(nd) Editions,Wiley-Interscience) and Ben-Ishai, Tetrahedron, 43, 439-450 (1987).

In Scheme 2, step 1, an appropriate phenyl acetic acid of formula (5) iscoupled with an appropriate acetal of formula (6) to give a compound offormula (7). An appropriate phenyl acetic acid of formula (5) is one inwhich A₂ is an activated group, for example, —OH, —Cl, or —Br. Anappropriate acetal of formula (6) is one in which R₅ is a C₁-C₄ alkyl.Such coupling reactions are common in peptide synthesis and syntheticmethods used therein can be employed as are described in Scheme 1, step1.

Also, the coupling depicted in Scheme 2, step 2, can be carried outunder Schotten-Baumann conditions using an acid halide of the compoundof formula (5) and an appropriate acetal of formula (6) in a mixedsolvent, such as, methyl t-butyl ether, ethyl acetate, tetrahydrofuran,acetone, or diethyl ether and water. Such reaction are carried out usinga suitable base, such as sodium hydroxide, potassium hydroxide, sodiumcarbonate, potassium carbonate, sodium bicarbonate, or potassiumbicarbonate. Typically the reaction is stirred or agitated vigorouslyand is carried out at temperatures of from about −20° C. to about 80° C.and typically require from about 1 to about 24 hours. Upon reactioncompletion, the product of formula (7) is recovered by conventionalmethods including extraction, precipitation, chromatography, filtration,trituration, crystallization and the like.

In Scheme 2, step 2, a compound of formula (7) is cyclized to give acompound of formula (8). Such cyclization reactions are carried out in aacid, such as sulfuric acid. Typically the acid is used as the solvent.In general, the reaction is carried out at temperatures of from about−20° C. to about 150° C. and typically require from about 1 to about 24hours. Upon reaction completion, the product of formula (8) is recoveredby conventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization and the like.

In Scheme 2, step 3, a compound of formula (8) undergoes an aminetransfer reaction to give a compound of formula (9). In Scheme 2 anoximation is depicted. Such oximation are accomplished by contacting theenolate of a compound of formula (8) with an oxime transfer reagents,such as an alkyl nitrite ester. The enolate of a compound of formula (8)can be prepared by reacting the compound of formula (8) with a suitablebase, such as potassium t-butoxide, lithium diisopropylamide, lithiumhexamethylsilazide, sodium hexamethylsilazide, potassiumhexamethylsilazide, and the like. Such oximinations are exemplified byWheeler, et al., Organic Syntheses, Coll. Vol. VI, p. 840 whichdescribes the reaction of isoamyl nitrite with a ketone to prepare thedesired oxime. The reaction is typically carried out in a solvent, suchas tetrahydrofuran. In general, the reaction is carried out attemperatures of from about −20° C. to about 50° C. and typically requirefrom about 1 to about 24 hours. Upon reaction completion, the product offormula (8) is recovered by conventional methods including extraction,precipitation, chromatography, filtration, trituration, crystallizationand the like.

Alternately, such an amine transfer reaction can be accomplished throughthe azide. An azide can be formed by the reaction of the enolate of acompound of formula (8) with an azide transfer reagent, such astoluenesulfonyl azide and triisopropylbenzenesulfonyl azide. Suchreaction are exemplified in Evans, et al., J. Am. Chem. Soc.,112:4011-4030 (1990)41. The reaction is typically carried out in asolvent, such as tetrahydrofuran. In general, the reaction is carriedout at temperatures of from about −20° C. to about 50° C. and typicallyrequire from about 1 to about 24 hours. Upon reaction completion, theproduct of formula (8) having an azide instead of an oxime is recoveredby conventional methods including extraction, precipitation,chromatography, filtration, trituration, crystallization and the like.

As depicted in Scheme 2, step 4, an oxime is reduced to the compound offormula (4). Such reductions are accoplished by treatment with hydrogenand a suitable catalsyt, such as Raney-nickel or palladium catalysts,such as palladium-on-carbon. The reaction is typically carried out in asolvent, such as tetrahydrofuran, ethyl acetate, or lower alcohols, suchas methonol, ethanol, and isopropanol, in acetic acid, water, aqueousammonia, and the like, and mixtures thereof. The reaction generallycarried out at hydrogen pressures ranging from atmospheric pressure toabout 600 psi (4137 kPa). In general, the reaction is carried out attemperatures of from about 20° C. to about 100° C. and typically requirefrom about 1 to about 24 hours. Upon reaction completion, the product offormula (4) is recovered by conventional methods including extraction,precipitation, chromatography, filtration, trituration, crystallizationand the like.

Alternately, where the amine is transferred via an azide, the azidogroup is reduced. Such reductions are carried out by hydrogenation asdescribed above.

Processes for making(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneare described in Scheme A.

Scheme A, step 1, depicts the stereochemical resolution of anappropriate lactam of formula (4) to give a lactam of formula (10), thatis, of a substantially pure (S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one. As used herein the term“substantially pure” refers to enantiomeric purity of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.Accordingly to the present invention substantially pure(S)-1-amino-3-methyl-4, 5,6,7-tetrahydro-2H-3-benzazepin-2-one can beprepared comprising the (S)-enantiomer which is greater than 80%,preferably greater than 90%, more preferably greater than 95%, mostpreferably greater than 97%.

For example, the (S)-isomer of the compound of formula (4) can beresolved by fractional crystallization of dibenzoyltartrate,(R)-(−)-d-camphorsulfonic acid, and (D)-(−)-mandelic acid salts. It isexpected that a wide variety of dibenzolytartarates are suitable forthis purpose. In particular, the dibenzoyl esters having a parasubstituent selected from the group consisting of hydrogen, halogen,C₁-C₄ alkyl, and C₁-C₄ alkoxy are preferred with di-p-toluoyl-tartratebeing preferred. Di-p-toluoyl-L-tartrate is used to obtain the(S)-isomer.

According to the present process, the compound of formula (4) iscontacted with the selected acid. Generally, from about 0.4 molarequivalents to a large excess of the selected acid can be used withabout 0.4 to 1.5 molar equivalents being preferred and with about 0.5 to1.1 molar equivalents being more preferred.

The process is typically carried out by crystallizing the acid additionsalt from a solution. In particular, solvents such as lower alcohols,including methanol, ethanol, n-propanol, isopropanol, butanol,sec-butanol, iso-butanol, t-butanol, amyl alcohol, iso-amyl alcohol,t-amyl alcohol, hexanol, cyclopentanol, and cyclohexanol are suitable,with methanol, ethanol, and isopropanol being preferred. The use of ananti-solvent may be advantageous. As used herein, the term“anti-solvent” refers to a solvent in which the salt is significantlyless soluble compared to solvent. Preferably, when an anti-solvent isused it is miscible with the selected solvent. Suitable anti-solventsinclude ethers, such as diethyl ether, methyl t-butyl ether, and thelike, and lower alkyl acetates, such as methyl acetate, ethyl acetate,iso-propyl acetate, propyl acetate, iso-butyl acetate, sec-butylacetate, butyl acetate, amyl acetate, iso-amyl acetate, and the like,and alkanes, such as pentane, hexane, heptane, cyclohexane, and thelike. When the present process is carried out by crystallizing the acidaddition salt from the racemic mixture, care must be taken in using ananti-solvent to avoid crystallization of the salt of the undesireddiastereomeric salt.

Typically, the crystallization is carried out at initial temperatures ofabout 40° C. to reflux temperature of the selected solvent(s) and atinitial concentrations of from about 0.05 molar to about 0.25 molar. Themixture is then cooled to give the salt. Seeding may be advantageous.Stirring of the initial precipitate for from about 4 to 48 hours may beadvantageous. Preferably the crystallization solution is cooled slowly.The crystallization is most conveniently cooled to temperatures ofambient temperature to about −20° C. The salt can be collected usingtechniques that are well known in the art, including filtration,decanting, centrifuging, evaporation, drying, and the like. The compoundof formula (10) can be used directly as the acid addition salt of theselected acid. Alternately, before use the compound of formula (10) canbe isolated as another acid addition salt after acid exchange or can byisolated as the base by extraction under basic conditions as is wellknown and appreciated in the art.

A preferred process gives(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one ofsubstantial enantiomeric purity by crystallizing1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one as its acidaddition salt of an acid selected from the group consisting ofdi-p-tolyl-L-tartaric acid, (R)-(−)-d-camphorsulfonic acid, and(D)-(−)-mandelic acid as a dynamic process in the presence of anaromatic aldehyde. The dynamic process has the advantage that the1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one undergoesconversion to a single isomer during the crystallization, thus,improving the yield and avoiding a waste stream which includes anundesired isomer.

It is expected that a wide variety of aromatic aldehydes are suitablefor the dynamic process, we have found that a number of aldehydes areparticularly suitable in practice. Specifically, we have found thatsalicylic acids are preferred and salicylaldehyde,5-nitrosalicylaldehyde, and 3,5-dichlorosalicylaldehyde are morepreferred in the present dynamic resolution process.

Accordingly, when the present process is carried out as a dynamicresolution, 1-amino-3-methyl-4, 5,6,7-tetrahydro-2H-3-benzazepin-2-oneis contacted with the selected acid in the presence of an aromaticaldehyde. Generally, for the dynamic resolution from about 0.9 to 1.2molar equivalents of acid are used, with about 1 molar equivalents beingpreferred. The aromatic aldehyde is generally used in a catalyticamount. Typically, about 0.5 to 0.001 molar equivalents of aromaticaldehyde are used, with about 0.1 to about 0.01 molar equivalents beingpreferred.

The dynamic process is typically carried out in a solvent without ananti-solvent as described above. The mixture of1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, the selectedacid, and aromatic aldehyde are stirred to allow conversion to thedesired isomer. Generally this conversion is carried out at temperaturesof from ambient temperature to the refluxing temperature of the solvent.Generally conversion requires 6 to 48 hours.

As will be appreciated by the skilled artisan, when the present processis carried out as a dynamic resolution, use of the acid addition salt of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one can becomplicated by the presence of a small amount of aromatic aldehyde inthe isolated product. Thus, after dynamic resolution it is preferredthat (S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one beisolated by salt exchange, preferably as the hydrochloride salt, beforeits use or formation of base.

Scheme A, step 2, depicts the coupling reaction of an appropriateamino-protected alanine of formula the PgNH—CHCH₃—C(O)-A and anappropriate lactam of formula (10). Appropriate amino-protected alanineis ones in which Pg is an amine protecting group, is of theL-configuration, and A is an activating group, for example —OH or —Cl,capable of coupling with the amino group of the compound of formula(10). Such amino-protected alanines are readily available to the personskilled in the art.

The coupling reaction depicted in Reaction Scheme A, step 2, involves areaction which is conventionally conducted for peptide synthesis andsynthetic methods used therein can also be employed. Such methods aredescribed in detail in Scheme 1, step 1.

Reaction Scheme A, step 3, depicts the deprotection of a compound offormula (11) to give a compound of formula (12). Such deprotections ofamino protecting groups is well known and appreciated in the art.

Reaction Scheme A, step 4, depicts the coupling reaction of anappropriate compound of formula (13), (CH₃)₂CH—CHOH—C(O)A₁ and acompound of formula (12) to give a compound of formula I. The S-isomerof the compound of formula (13) is commercially available and is wellknown in the art, including PCT Application No. PCT/US97/22986, filed 22Dec. 1997. The coupling reaction depicted in step 3 is carried out usingthe acid of formula (13) (compounds in which A₁ is —OH) or the acidhalide derived therefrom (compounds in which A₁ is —Cl or —Br), in amanner similar to those taught in Scheme 1, step 1.

An alternative method for preparing the compounds of formula I isdepicted in Scheme A, step 5, which shows the coupling reaction of anappropriate compound of formula (10) and an appropriate compound offormula (14), (CH₃)₂CH—CHOH—C(O)—NH—CHCH₃—C(O)A₂, to directly give acompound or formula I. An appropriate compound of formula (10) is asdescribed in step 2. An appropriate compound of formula (14) is one inwhich has the stereochemistry as desired in the final product of formulaI.

Compounds of formula (14) are readily prepared by couplingcarboxy-protected amino acids, H₂N—CHCH₃—C(O)OPg₁, with compounds offormula (13) as described above. Again such coupling reactions are wellknown in the art and afford a product, which after deprotection,provides a compound of formula (14).

The compound of formula I can be isolated and purified by a number oftechniques, including crystallization. Crystallization from a solutionand slurrying techniques are can be used. In particular, the compound ofthe present invention can be prepared by crystallization from a varietyof anhydrous and aqueous solvents. Suitable solvents acetone, loweralcohols (like methanol, ethanol, and isopropanol), acetic acid, andacetonitrile with and without water and ethyl acetate, diethyl ether,and methyl t-butyl ether. In practice, it has been found that aqueousacetone is preferred. For a given aqueous solvent the amount of waterused will depend on the relative solubility of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onein the solvent compared to water and whether a crystallization orslurrying technique is used.

A crystallization is generally carried out by dissolving,(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onein an aqueous solvent and then allowing the solution to cool, with orwithout the addition of more water, to give a solid. Typically, thecrystallization is carried out at initial temperatures of about 40° C.to reflux temperature of the selected aqueous solvent. The mixture isthen-cooled to give the crystalline dihydrate. Seeding may beadvantageous. Preferably the crystallization solution is cooled slowly.The crystallization is most conveniently cooled to temperatures ofambient temperature to about −20° C.

The present invention is further illustrated by the following examplesand preparations. These examples and preparations are illustrative onlyand are not intended to limit the invention in any way.

The terms used in the examples and preparations have their normalmeanings unless otherwise designated. For example “°C” refers to degreesCelsius; “mmol” refers to millimole or millimoles; “g” refers to gram orgrams; “mL” refers milliliter or milliliters; “brine” refers to asaturated aqueous sodium chloride solution; “THF” refers totetrahydrofuran; “HPLC” refers to high pressure liquid chromatography;etc.

EXAMPLE 1 Synthesis of1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

To a slurry of sodium hydride (1.1 eq) in 15 mL of dry DMF was added4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (0.0042 moles) as a solution in10 mL of DMF. Methyl iodide (about 2 eq.) was then added. When completeby TLC, the reaction mixture was poured over ice and extracted intoethyl acetate. The organic layer was washed with water, followed bybrine. The organic layer was then dried over Na₂SO₄, filtered, andconcentrated under reduced pressure. The residue was purified by HPLC(LC 2000), eluting with an ethyl acetate/hexane system to give3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.

3-Methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (1 eq.) was dissolvedin THF and isoamylnitrite (1.2 eq.) was added. The mixture was cooled to0° C. in an ice bath. NaHMDS (1.1 eq., 1M in THF) was added dropwise.After stirring for 1 hour or until the reaction was complete, themixture was concentrated then acidified with 1N aqueous hydrochloricacid solution and extracted with ethyl acetate. The organic portion wasdried and concentrated to yield a crude product which was purified bysilica gel chromatography to give1-hydroxyimino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one: Massspectroscopy (M+H)⁺, 205.1.

1-Hydroxyimino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one wasdissolved in EtOH/NH₃ (20:1) and hydrogenated in a bomb using Raneynickel and hydrogen (500 psi/3447 kPa) at 100° C. for 10 hours. Theresulting mixture was filtered and concentrated to provide an oil whichwas purified by silica gel chromatography to yield the title compound.

EXAMPLE 2 Synthesis of1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

To a 20 L Morton flask was added MTBE (5.52 L, 7 volumes) and(N-methylamino)-acetaldehyde dimethyl acetal (614 g, 5 mol) to form asolution at room temperature. A solution of sodium bicarbonate preparedby the addition of sodium bicarbonate (546 g, 6.5 mol) and water (6.31L, 8 volume) was added to the Morton reaction flask. The mixture wascooled to less than 10° C. and a MTBE (789 mL) solution of phenylacetylchloride (789 g, 5 mol) was added dropwise to the cooled reactionmixture over a 1 h period. After addition, the reaction mixture wasstirred at room temperature for 1 h. At this stage an HPLC analysisindicated that the reaction was completed. Extractive workup with MTBE(4 volumes), anhydrous magnesium sulfate drying followed byconcentration on the rotary evaporator provided 1.187 kg (98%) ofN-methyl-N-(2,2-dimethoxyethyl)phenylacetamide as a liquid,(M+H)⁺=237.9. To a 5 L Morton flask under a strong nitrogen atmospherewas added H₂SO₄, (1.42 L) andN-methyl-N-(2,2-dimethoxyethyl)phenylacetamide (712 g, 3 mol) was addeddropwise to the reaction flask which caused an exotherm (22 to 78° C.).The resulting reaction was then heated to 110° C. for 3 h then cooled toroom temperature and transferred to a 20 L Morton flask. At less than10° C., the reaction mixture was quenched with aqueous sodium hydroxide(9.18 L, 5 N). Extractive workup with ethyl acetate (2×2.85 L), dryingwith sodium sulfate followed by concentrating to a solid, provided 520 g(73.5%) of 3-methyl-6,7-dihydro-2H-3-benzazepin-2-one as a solid. Thismaterial may be recrystallized from MTBE for added purity to give asolid, mp=81-82° C.; (M+H)⁺=174.2.

A THF (0.5 L) solution of 3-methyl-6,7-dihydro-2H-3-benzazepin-2-one(113.8 g, 0.657 mol) was cooled to 0° C. and isoamyl nitrite (100.75 g,0.86 mol) was added dropwise. To the resulting mixture was added LiHMDS(1 N THF solution, 854 mL, 0.854 mol) at a rate such that thetemperature remained below 10° C. After addition, the reaction wasallowed to stir at room temperature for 2-3 h while monitoring for thereaction progress by HPLC. Upon completion of the reaction, the mixturewas cooled to 0° C., and the pH adjusted from 12 to 2-3 using aqueousHCl (2N). The resulting precipitate was stirred for 12-16 h beforeisolation by filtration and drying to provide 86.3 g (64.9%) of1-hydroxyimino-3-methyl-6,7-dihydro-2H-3-benzazepin-2-one; mp=225-226°C.; (M+H)⁺=203.0.

An ethanol (525 mL) solution of1-hydroxyimino-3-methyl-6,7-dihydro-2H-3-benzazepin-2-one (35 g, 0.173mol) was added to an autoclave along with palladium on carbon (10%, 3.5g) as a dilute HCl (concentrated aqueous, 17.5 g in 17 mL water) slurry.The resulting mixture was hydrogenated at 50° C. and 250 psi (1723 kPa)until the reaction was completed. The reaction mixture was filtered overa pad of celite using ethanol as solvent and the filtrate concentratedto 90 mL. Water (350 mL) was added to the concentrate and the resultingsolution further concentrated to about 200 mL. Dichloromethane (350 mL)was added to the aqueous solution before adjusting the pH to 11-11.5with aqueous sodium hydroxide (1 N). The organic portion was separatedand the aqueous portion extracted with dichloromethane (175 mL). Thecombined extracts were concentrated to a residue that crystallized uponstanding to give the title compound: mp=69-81° C.; (M+H)⁺=191.0.

EXAMPLE 3 Synthesis of1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

To a 22 L Morton flask was added dichloromethane (4.73 L, 8 volumes),N-methylphenethylamine (591 g, 4.33 mol), and aqueous sodium bicarbonate(436.7 g, 5.2 mol in 4.73 L of water). The mixture was cooled to lessthan 5° C. and dichloromethane (887 mL) solution of chloroacetylchloride (513.7 g, 4.55 mol) was added dropwise to the cooled reactionmixture over a 70 min period. After addition, an HPLC analysis indicatedthat the reaction was completed. The layers were separated and theaqueous layer was extracted with dichloromethane. Combined organiclayers were dried over anhydrous magnesium sulfate and concentrated onthe rotary evaporator to provide 915.7 g (99.8%) ofN-methyl-N-(2-phenylethyl)-1-chloroacetamide: (M+H)=212.1.

To a 12 L flask under a nitrogen atmosphere was addedN-methyl-N-(2-phenylethyl)-1-chlbroacetamide (883.3 g, 4.17 mol) andortho-dichlorobenzene (6.18 L). Add aluminum chloride (1319 g, 10.13mol) which caused an exotherm (22 to 50° C.). The resulting reaction wasthen heated to 165° C. for 2.5 h then cooled to room temperature overabout 14 hours. The reaction mixture was cooled to about 0° C., and wasadded to cold water (8.86 L, about 5° C.) in four portions in order tokeep exotherm to about 40° C. The layers were separated and aqueouslayer was extracted with dichloromethane (7.07 L) and the layersseparated. The organic layers were combined and extracted with aqueoushydrochloric acid (8.83 L, 1N) and then a saturated aqueous sodiumbicarbonate solution (7.07 L), dried over magnesium sulfate, combinedwith silica gel (883 g) and applied to a column of silica gel (3.53 kg,in a sintered glass funnel, packed as a slurry in dichloromethane). Thecolumn was eluted with dichloromethane until 25 L were collected andthen with ethyl acetate to provide the product. The product containingfraction were evaporated to3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one as a tan solid, 608 g(83%).

In a 22 L flask, under nitrogen, was3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (606 g, 3.46 mol) andisoamyl nitrite (543 g, 4.5 mol) in THF (7.88 L). The mixture was cooledto about 0° C. before LiHMDS (1 N THF solution, 4.5 L, 04.5 mol) wasadded at a rate such that the temperature remained below about 7° C.After addition, the reaction was allowed to stir at room temperature forabout 2 h while monitoring for the reaction progress by HPLC. Uponcompletion of the reaction, the mixture was cooled to about 0° C., andthe pH adjusted from 12 to about 2-1 using aqueous HCl (2N). Theresulting precipitate was stirred for about 6 h before isolation byfiltration and drying to provide1-hydroxyimino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one 604.7 g(85.6%).

1-Hydroxyimino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (625 g,3.06 mol) and 3A ethanol (15.6 L), The resulting mixture washydrogenated at 50° C. and 250 psi (1723 kPa) with vigorous agitationuntil the reaction was completed (about 4 hours). The reaction mixturewas filtered over a pad of celite using ethanol as solvent and thefiltrate concentrated give a solid. The solid was treated withdichloromethane (6 L) and 1N aqueous sodium hydroxide solution was addeduntil the pH to of the aqueous layer was between 11-11.5. The mixturewas agitated, the layers were separated, and the aqueous layer wasextracted with dichloromethane (2 L). The organic layers were dried overmagnesium sulfate, filtered, and evaporated in a rotary evaporator togive the title compound 477 g (81.9%).

EXAMPLE 4 Synthesis of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

1-Amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (1.544 g, 8.12mmol) was heated gently in 15 mL methanol to form a solution. In anotherflask, di-p-toluoyl-1-tartaric acid (3.12 g, 8.08 mmol) was dissolved in15 mL methanol and added via pipette to the warm amine solution. Themixture was heated as solids precipitated. An additional 30 mL ofmethanol was added to achieve a solution, which was refluxed for 30-40minutes and then slowly cooled to ambient temperature to give a solid.After stirring for about 18 hours, the solid was collected by filtrationand rinsed with a small amount of cold methanol to give 2.24 g of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onedi-p-toluoyl-L-tartaric acid salt (96% yield, 94.7% ee).

(S)-1-Amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onedi-p-toluoyl-L-tartaric acid salt (11.83 g, 20.5 mmol) was dissolved in45 mL of aqueous 1.0 N sodium hydroxide solution and extracted withmethylene chloride (3×25 mL). The combined methylene chloride layerswere washed with 35 mL aqueous 1.0 N sodium hydroxide solution, thenbrine solution, and dried over anhydrous MgSO₄. Removal of solvent undervacuum gave the title compound (3.38 g) as a colorless oil (87% yield,93.2% ee).

EXAMPLE 5 Synthesis of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

1-Amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (6.0 g, 31.5mmol) was heated gently in 75 mL methanol to form a solution andcombined with a solution of di-p-toluoyl-L-tartaric acid (12.2 g, 31.5mmol) in 75 mL of warm methanol. The solution was seeded and a solidformed. An additional 100 mL of methanol was added and the mixture wasallowed to stir. After stirring for about 18 hours, the solid wascollected by filtration and rinsed with a small amount of cold methanolto give 6.7 g of a solid. The solid was combined with methanol (200 mL),and stirred. After 18 hours, the solid was collected to give(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onedi-p-toluoyl-L-tartaric acid salt (4.4 g). Isolation of the base by theprocedure described in Example 4 gave the title compound (96% ee).

EXAMPLE 6 Synthesis of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

In a 22 L vessel, under nitrogen,1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (438 g, 2.3mol) was heated (about 40° C.) to provide a solution in methanol (4.38mL). In another flask, di-p-toluoyl-1-tartaric acid (889.7 g, 2.3 mol)was dissolved in 4.38 L of methanol and heated to about 40° C. beforethe solution of1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one was added. Theheating was continued and an additional 6.13 L of methanol was addedbefore the mixture was refluxed for about 45 minutes and then slowlycooled to ambient temperature to give a solid. After stirring for about18 hours, the solid was collected by filtration and rinsed with a smallamount of mother liquors, and after air drying, with about 2 L of ethylacetate to give 561.6 g of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onedi-p-toluoyl-L-tartaric acid salt. Combine(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onedi-p-toluoyl-L-tartaric acid salt, dichloromethane (6.57 L) and 1Naqueous sodium hydroxide solution (6.57 L) and agitate. Separate thelayers and extract the organic layer twice with and 1N aqueous sodiumhydroxide solution (3.28 L), once with brine (2.46 L) before drying overmagnesium sulfate, filtering, and evaporating on a rotary evaporator togive the title compound 250 g (57.4%, 94.1% ee).

EXAMPLE 7 Synthesis of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onehydrochloric acid salt

1-Amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (31.9 g, 168mmol) was slurried in about 300 mL isopropyl acetate and heated to 45°C. In a separate flask, (R)-(−)-D-mandelic acid (25.0 g, 164 mmol) washeated in about 130 mL of isopropyl alcohol until a solution formed andwas added to the1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one/isopropylacetate slurry obtained above to give a solution from which aprecipitate quickly formed. The mixture was stirred at 45° C. for about3 hours. 5-Nitrosalicylaldehyde (2-hydroxy-5-nitrobenzaldehyde) (1.40 g,8.38 mmol, 5 mol %) was added to the warm solution and the mixture wasstirred at 45° C. After about 14 hours, the slurry was cooled to ambienttemperature and stirred for 2 hours before the solids were collected byfiltration and rinsed with 70 mL of cold isopropyl acetate, and dried inthe vacuum oven at 40° C. to obtain 46.62 g of(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one(R)-mandelic acid salt (82.9% yield, 98.4% ee).

(S)-1-Amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one(R)-mandelic acid salt (2.42 g, 7.06 mmol, 98.4% ee) was slurried in 25mL ethyl acetate at ambient temperature. Concentrated aqueoushydrochloric acid (1.1 mL, about 11.2 mmol) was added and the mixturewas heated to 50° C. with vigorous stirring for 3.5 hours. The slurrywas cooled to ambient temperature and filtered, rinsed with the methylt-butyl ether (about 10 mL) to give 1.48 g of the title compound (92.5%yield, 97.9% ee).

EXAMPLE 8 Synthesis of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

A round bottom flask was charged with N-t-Boc-L-alanine (1.0 eq.),hydroxybenzotriazole hydrate (about 1.1 eq.) and(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (1.0 eq.)in THF under nitrogen atmosphere. Hunig's base(N,N-diisopropylethylamine, 1.1 eq.) was added to the well stirredmixture followed by EDC (1.1 eq.). After stirring from 4 to 17 hours atambient temperature the solvent was removed at reduced pressure, theresidue taken up in ethyl acetate and water, washed with saturatedaqueous sodium bicarbonate solution, 1 N aqueous HCl, brine, dried overanhydrous sodium sulfate, filtered, and the solvent removed at reducedpressure to provide1-(N-t-Boc-L-alaninyl)amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one:mass spectroscopy (M+H)⁺, 362.3.

A stream of anhydrous HCl gas was passed through a stirred solution of1-(N-t-Boc-L-alaninyl)amino-3-methyl-4,5,6,7-tetrahydro-ZH-3-benzazepin-2-onein 1,4-dioxane(0.03-0.09 M), chilled in a ice bath to about 10° C. underNZ, for 10-15 minutes. The solution was capped, then the cooling bathremoved, and the solution was allowed to warm to ambient temperaturewith stirring for 2-8 hours, monitoring 1.0 by TLC for the consumptionof starting material. The solution was concentrated to give1-(L-alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onewhich was used without further purification.

1-(L-Alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-3-methyl-2H-3-benzazepin-2-one(1.0 eq.), hydroxybenzotriazole hydrate (1.1 eq.) and(S)-2-hydroxy-3-methyl-butyric acid (1.0 eq.) in THF under nitrogenatmosphere. Hunig's base (N,N-diisopropylethylamine, 1.1 eq.) was addedto the well stirred mixture followed by EDC (1.1 eq.). After stirringfrom 4 to 17 hours at ambient temperature the solvent was removed atreduced pressure, the residue taken up in ethyl acetate (or similarsolvent) and water, washed with saturated aqueous sodium bicarbonatesolution, 1 N HCl, brine, dried over anhydrous sodium sulfate and thesolvent removed at reduced pressure to provide the title compound.

EXAMPLE 9 Synthesis of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one

A round bottom flask was charged with N-t-Boc-L-alanine (249.5 g, 1.32mol), hydroxybenzotriazole hydrate (232.2 g, 1.52 mol), and(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one (250.8 g,1.32 mol) in THF (3.76 L) under nitrogen atmosphere. The mixture wascooled to less than 5° C. before adding Hunig's base(N,N-diisopropylethylamine, 188.4 g, 1.45 mol) followed by EDC (283.7 g,1.45 mol). After stirring 6 hours the reaction mixture was warmed toambient temperature and stirred for about 14 hours. The solvent wasremoved at reduced pressure, the residue taken up in ethyl acetate (3.76L) and water (1.76 L), the layers were separated, the organic layerextracted with water (1.76 L), the aqueous layers combined and extractedwith ethyl acetate (1.76 L). The organic layers were combined, extractedwith saturated aqueous sodium bicarbonate solution (1.76 L), dried overanhydrous sodium sulfate, filtered, and evaporated in on a rotaryevaporator to provide1-(N-t-Boc-L-alaninyl)amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one463 g (97.2%).

An ethyl acetate solution of HCl was prepared by passing anhydrous HClgas, using a subsurface dispersion tube, through ethyl acetate (1.76 L)cooled to about 0° C. The ethyl acetate solution of HCl prepared abovewas added to a vigorously stirred slurry of1-(N-t-Boc-L-alaninyl)amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one(462 g, 1.28 mol) in ethyl acetate (3.7 L). An additional amount ofethyl acetate (1 L) was added and the reaction mixture was allowed towarm to room temperature and stirred for 22 h. The reaction mixture wasfiltered to give a solid. The solid was slurryed with acetonitrile (5L), heated to relux and then cooled to about 60° C. before filtering anddrying to give1-(L-alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one389.8 g (94.7%).

1-(L-Alaninyl)-(S)-amino-3-methyl-4,5,6,7-tetrahydro-3-methyl-2H-3-benzazepin-2-one(369.5 g, 1.18 mol), hydroxybenzotriazole hydrate (207.6 g, 1.36 mol),Hunig's base (N,N-diisopropylethylamine, 352.2 g, 2.71 mol), and(S)-2-hydroxy-3-methyl-butyric acid (140.6 g, 1.18 mol) in THF (4.8 L)were combined under a nitrogen atmosphere and cooled to less than 5° C.EDC (253.7 g, 1.3 mol) was added and the reaction mixture was allowed towarm to ambient temperature and to stir. After about 25 hours thereaction mixture was diluted with dichloromethane (5.54 L) and extractedwith water (2.22 L). The organic layer was extracted with water (2.22L), the aqueous layers were combined and extracted with dichloromethane(5.54 L). The organic layers were combined, extracted twice with water(2.22 L), with saturated aqueous sodium bicarbonate solution (2.22 L),dried over anhydrous sodium sulfate, filtered, and evaporated in on arotary evaporator to provide a solid 428 g (100%). The solid was takenup in a solvent mixture containing acetone (3.42 L) and water (0.856 L)with slight warming (40° C.). The solution was split into ˜2 L portionsand to each was added water (7.19 L) while warming the hazy solution to50° C. Upon complete addition of water the hazy solution was allowed tocool to ambient to give a solid which was stirred as a slurry at ambienttemperature for about 14 hours before filtering and drying to give thetitle compound 310.6 g (66.2%) as its dihydrate.

When employed as a pharmaceutical the present invention is usuallyadministered in the form of a pharmaceutical composition. Thus, inanother embodiment, the present invention provides pharmaceuticalcompositions comprising an effective amount ofN-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneand a pharmaceutically acceptable diluent. Such compositions are usedfor inhibiting β-amyloid peptide release and/or its synthesis, includingthe treatment of Alzheimer' disease. Thus, the present inventionencompasses the use of-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-onefor the manufacture of a medicament for inhibiting β-amyloid peptiderelease and/or its synthesis, and specifically including, treatingAlzheimer's disease.

(N)-((S)-2-Hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one can beadministered by a variety of routes. The present compound can beadministered in any form or mode which makes the compound bioavailablein an effective amount, including oral and parenteral routes. Forexample, the present compound can be administered orally, by inhalation,subcutaneously, intramuscularly, intravenously, transdermally,intranasally, rectally, occularly, topically, sublingually, buccally,and the like.

In making the compositions of this invention, the active ingredient isusually mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier which can be in the form of a capsule, sachet,paper or other container. The compound of the present invention can beadministered alone or in the form of a pharmaceutical composition, thatis, combined with pharmaceutically acceptable diluents, such as carriersor excipients, the proportion and nature of which are determined by thesolubility and chemical properties of the present compound, the chosenroute of administration, and standard pharmaceutical practice.(Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co.(1990)).

The present pharmaceutical compositions are prepared in a manner wellknown in the pharmaceutical art. The carrier or excipient may be asolid, semi-solid, or liquid material which can serve as a vehicle ormedium for the active ingredient. Suitable carriers or excipients arewell known in the art. The pharmaceutical composition may be adapted fororal, inhalation, parenteral, or topical use and may be administered tothe patient in the form of tablets, capsules, aerosols, inhalants,suppositories, solution, suspensions, or the like.

For the purpose of oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gums and thelike. These preparations should contain at least 4% of the compound ofthe present invention, the active ingredient, but may be varieddepending upon the particular form and may conveniently be between 2% toabout 90% of the weight of the unit. The amount of the compound presentin compositions is such that a suitable dosage will be obtained.Preferred compositions and preparations according to the presentinvention may be determined by a person skilled in the art.

The tablets, pills, capsules, troches, and the like may also contain oneor more of the following adjuvants: binders such as microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch orlactose, disintegrants such as alginic acid, Primogel, corn starch andthe like; lubricants such as magnesium stearate, silicon oil, orSterotex; glidants such as colloidal silicon dioxide; and sweeteningagents such as sucrose or saccharin may be added or a flavoring agentsuch as peppermint, methyl salicylate or orange flavoring. When thedosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as polyethylene glycol or afatty oil. Other dosage unit forms may contain other various materialswhich modify the physical form of the dosage unit, for example, ascoatings. Thus, tablets or pills may be coated with sugar, shellac, orother coating agents. A syrup may contain, in addition to the presentcompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors. Materials used in preparing these variouscompositions should be pharmaceutically pure and non-toxic in theamounts used.

For the purpose of parenteral administration, the compound of thepresent invention may be incorporated into a solution or suspension.These preparations typically contain at least 0.1% of the compound ofthe invention, but may be varied to be between 0.1 and about 90% of theweight thereof. The amount of the compound present in such compositionsis such that a suitable dosage will be obtained. The solutions orsuspensions may also include one or more of the following adjuvants:sterile diluents such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl paraben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylene diaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Preferred compositions and preparations areable to be determined by one skilled in the art.

The compound of the present invention may also be administeredtopically, and when done so the carrier may suitably comprise asolution, ointment, or gel base. The base, for example, may comprise oneor more of the following: petrolatum, lanolin, polyethylene glycols,bees wax, mineral oil, diluents such as water and alcohol, andemulsifiers, and stabilizers. Topical formulations may contain aconcentration of the formula I or its pharmaceutical salt from about 0.1to about 10% w/v (weight per unit volume).

Another preferred formulation of the present invention employstransdermal delivery devices (“patches”). Such transdermal patches maybe used to provide continuous or discontinuous infusion of the compoundof the present invention in controlled amounts. The construction and useof transdermal patches for the delivery of pharmaceutical agents is wellknown in the art. See, e.g., U.S. Pat. No. 5,023,252, issued Jun. 11,1991, herein incorporated by reference. Such patches may be constructedfor continuous, pulsatile, or on demand delivery of pharmaceuticalagents.

In order to more fully illustrate the operation of this invention,typical pharmaceutical compositions are described below. The examplesare illustrative only, and are not intended to limit the scope of theinvention in any way.

FORMULATION EXAMPLE 1

Hard gelatin capsules containing the following ingredients are prepared:Ingredient Quantity(mg/capsule) Active Ingredient 30.0 Starch 305.0Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in340 mg quantities.

FORMULATION EXAMPLE 2

A tablet formula is prepared using the ingredients below: IngredientQuantity(mg/tablet) Active Ingredient 25.0 Cellulose, microcrystalline200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing240 mg.

FORMULATION EXAMPLE 3

A dry powder inhaler formulation is prepared containing the followingcomponents: Ingredient Weight % Active Ingredient 5 Lactose 95

The active ingredient is mixed with the lactose and the mixture is addedto a dry powder inhaling appliance.

FORMULATION EXAMPLE 4

Tablets, each containing 30 mg of active ingredient, are prepared asfollows: Ingredient Quantity(mg/tablet) Active Ingredient 30.0 mg Starch45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone  4.0 mg(as 10% solution in sterile water) Sodium carboxymethyl starch  4.5 mgMagnesium stearate  0.5 mg Talc  1.0 mg Total  120 mg

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinyl-pyrrolidone is mixed with the resultant powders, which arethen passed through a 16 mesh U.S. sieve. The granules so produced aredried at 50° to 60° C. and passed through a 16 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 30 mesh U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yield tabletseach weighing 150 mg.

FORMULATION EXAMPLE 5

Capsules, each containing 40 mg of medicament are made as follows:Ingredient Quantity(mg/capsule) Active Ingredient  40.0 mg Starch 109.0mg Magnesium stearate  1.0 mg Total 150.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 150 mg quantities.

FORMULATION EXAMPLE 6

Suppositories, each containing 25 mg of active ingredient are made asfollows: Ingredient Amount Active Ingredient   25 mg Saturated fattyacid glycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

FORMULATION EXAMPLE 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose aremade as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose(89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Colorq.v. Purified water to  5.0 ml

The active ingredient, sucrose and xanthan gum are blended, passedthrough a No. 10 mesh U.S. sieve, and then mixed with a previously madesolution of the microcrystalline cellulose and sodium carboxymethylcellulose in water. The sodium benzoate, flavor, and color are dilutedwith some of the water and added with stirring. Sufficient water is thenadded to produce the required volume.

FORMULATION EXAMPLE 8

Capsules, each containing 15 mg of medicament are made as follows:Ingredient Quantity(mg/capsule) Active Ingredient  15.0 mg Starch 407.0mg Magnesium stearate  3.0 mg Total 425.0 mg

The active ingredient, starch, and magnesium stearate are blended,passed through a No. 20 mesh U.S. sieve, and filled into hard gelatincapsules in 560 mg quantities.

FORMULATION EXAMPLE 9

A subcutaneous formulation may be prepared as follows: IngredientQuantity Active Ingredient 1.0 mg corn oil   1 ml

Depending on the solubility of the active ingredient in corn oil, up toabout 5.0 mg or more of the active ingredient may be employed in thisformulation, if desired).

FORMULATION EXAMPLE 10

A topical formulation may be prepared as follows: Ingredient QuantityActive Ingredient 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g WhiteSoft Paraffin to 100 g

The white soft paraffin is heated until molten. The liquid paraffin andemulsifying wax are incorporated and stirred until dissolved. The activeingredient is added and stirring is continued until dispersed. Themixture is then cooled until solid.

In one of its method aspects, this invention is directed to a method forinhibiting β-amyloid peptide release and/or its synthesis comprisingadministering to a patient in need thereof with an effective amount of-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.In a particular method embodiment, the present invention provides amethod for treating Alzheimer's disease comprising administering to apatient in need thereof with an effective amount of(N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.

It is also recognized that one skilled in the art may affect theAlzheimer's disease by treating a patient presently afflicted with thedisease or by prophylactically treating a patient at risk to develop thedisease. Thus, the terms “treatment” and “treating” are intended torefer to all processes wherein there may be a slowing, interrupting,arresting, controlling, or stopping of the progression of Alzheimer'sdisease, but does not necessarily indicate a total elimination of allsymptoms. As such, the present methods include for preventing the onsetof Alzheimer's disease in a patient at risk for developing Alzheimer'sdisease, inhibiting the progression of Alzheimer's disease, andtreatment of advanced Alzheimer's disease.

As used herein, the term “patients refers to a warm blooded animal, suchas a mammal, which is afflicted with a disorder associated with increaseβ-amyloid peptide release and/or its synthesis, including Alzheimer'sdisease. It is understood that guinea pigs, dogs, cats, rats, mice,horses, cattle, sheep, and humans are examples of animals within thescope of the meaning of the term. Patients in need of such treatment arereadily diagnosed.

As used herein, the term “effective amount” of a compound of formula Irefers to an amount which is effective in inhibiting β-amyloid peptiderelease and/or its synthesis, and specifically, in treating Alzheimer'sdisease.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of conventionaltechniques and by observing results obtained under analogouscircumstances. In determining an effective amount, the dose ofN-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one,a number of factors are considered by the attending diagnostician,including, but not limited to: the potency and characteristics ofN-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one;the species of patient; its size, age, and general health; the degree ofinvolvement or the severity of the disease; the response of theindividual patient; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of other concomitant medication; and other relevantcircumstances.

An effective amount ofN-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneis expected to vary from about 0.1 milligram per kilogram of body weightper day (mg/kg/day) to about 100 mg/kg/day. Preferred amounts are ableto be determined by one skilled in the art.

tetrahydro-2H-3-benzazepin-2-one anhydrate is expected to vary fromabout 0.1 milligram per kilogram of body weight per day (mg/kg/day) toabout 100 mg/kg/day. Preferred amounts are able to be determined by oneskilled in the art.

TheN-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-oneanhydrate of the present invention can be tested in various biologicalsystems including the following.

EXAMPLE A

Cellular Screen for the Detection of Inhibitors of β-Amyloid Production

Numerous compounds of formula I above were assayed for their ability toinhibit β-amyloid production in a cell line possessing the Swedishmutation. This screening assay employed cells (K293=human kidney cellline) which were stably transfected with the gene for amyloid precursorprotein 751 (APP751) containing the double mutation Lys₆₅₁Met₆₅₂ toAsn₆₅₁Leu₆₅₂ (APP751 numbering) in the manner described in InternationalPatent Application Publication No. 94/10569⁸ and Citron et al.¹². Thismutation is commonly called the Swedish mutation and the cells,designated as “293 751 SWE”, were plated in Corning 96-well plates at2-4×10⁴ cells per well in Dulbecco's minimal essential media (Sigma, St.Louis, Mo.) plus 10% fetal bovine serum. Cell number is important inorder to achieve β-amyloid ELISA results within the linear range of theassay (−0.2 to 2.5 ng per mL).

Following overnight incubation at 37° C. in an incubator equilibratedwith 10% carbon dioxide, media were removed and replaced with 200 □L ofa compound of formula I (drug) containing media per well for a two hourpretreatment period and cells were incubated as above. Drug stocks wereprepared in 100% dimethyl sulfoxide such that at the final drugconcentration used in the treatment, the concentration of dimethylsulfoxide did not exceed 0.5% and, in fact, usually equaled 0.1%.

At the end of the pretreatment period, the media were again removed andreplaced with fresh drug containing media as above and cells wereincubated for an additional two hours. After treatment, plates werecentrifuged in a Beckman GPR at 1200 rpm for five minutes at roomtemperature to pellet cellular debris from the conditioned media. Fromeach well, 100 μL of conditioned media or appropriate dilutions thereofwere transferred into an ELISA plate precoated with antibody 266 [P.Seubert, Nature (1992) 359:325-327] against amino acids 13-28 ofβ-amyloid peptide as described in International Patent ApplicationPublication No. 94/10569⁸ and stored at 4° C. overnight. An ELISA assayemploying labeled antibody 3D6 [P. Seubert, Nature (1992) 359:325-327]against amino acids 1-5 of β-amyloid-peptide was run the next day tomeasure the amount of β-amyloid peptide produced.

Cytotoxic effects of the compounds were measured by a modification ofthe method of Hansen, et al. To the cells remaining in the tissueculture plate was added 25 μL of a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)(Sigma, St. Louis, Mo.) stock solution (5 mg/mL) to a finalconcentration of 1 mg/mL. Cells were incubated at 37 □C for one hour,and cellular activity was stopped by the addition of an equal volume ofMTT lysis buffer (20% w/v sodium dodecylsulfate in 50%dimethylformamide, pH 4.7). Complete extraction was achieved byovernight shaking at room temperature. The difference in the OD_(562 nm)and the OD_(650 nm) was measured in a Molecular Device's UV_(max)microplate reader as an indicator of the cellular viability.

The results of the β-amyloid peptide ELISA were fit to a standard curveand expressed as ng/mL β-amyloid peptide. In order to normalize forcytotoxicity, these results were divided by the MTT results andexpressed as a percentage of the results from a drug free control. Allresults are the mean and standard deviation of at least six replicateassays.

EXAMPLE B

In Vivo Suppression of β-Amyloid Release and/or Synthesis

This example illustrates how the compounds of this invention could betested for in vivo suppression of β-amyloid release and/or synthesis.For these experiments, 3 to 4 month old PDAPP mice are used [Games etal., (1995) Nature 373:523-527]. Depending upon which compound is beingtested, the compound is usually formulated at between 1 and 10 mg/mL.Because of the low solubility factors of the compounds, they may beformulated with various vehicles, such as corn oil (Safeway, South SanFrancisco, Calif.); 10% ethanol in corn oil;2-hydroxypropyl-β-cyclodextrin (Research Biochemicals International,Natick Mass.); and carboxy-methyl-cellulose (Sigma Chemical Co., St.Louis Mo.).

The mice are dosed subcutaneously with a 26 gauge needle and 3 hourslater the animals are euthanized via CO₂ narcosis and blood is taken bycardiac puncture using a 1 cc 25G ⅝” tuberculin syringe/needle coatedwith solution of 0.5 M EDTA, pH 8.0. The blood is placed in aBecton-Dickinson vacutainer tube containing EDTA and spun down for 15minutes at 1500×g at 5° C. The brains of the mice are then removed andthe cortex and hippocampus are dissected out and placed on ice.

1. Brain Assay

To prepare hippocampal and cortical tissue for enzyme-linkedimmunosorbent assays (ELISAs) each brain region is homogenized in 10volumes of ice cold guanidine buffer (5.0 M guanidine-HCl, 50 mMTris-HCl, pH 8.0) using a Kontes motorized pestle (Fisher, PittsburghPa.). The homogenates are gently rocked on a rotating platform for threeto four hours at room temperature and stored at −20° C. prior toquantitation of β-amyloid.

The brain homogenates are diluted 1:10 with ice-cold casein buffer[0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20μg/ml aprotinin, 5 mM EDTA, pH 8.0, 10 μg/ml leupeptin], therebyreducing the final concentration of guanidine to 0.5 M, beforecentrifugation at 16,000×g for 20 minutes at 4° C. Samples are furtherdiluted, if necessary, to achieve an optimal range for the ELISAmeasurements by the addition of casein buffer with 0.5 M guanidinehydrochloride added. The β-amyloid standards (1-40 or 1-42 amino acids)were prepared such that the final composition equaled 0.5 M guanidine inthe presence of 0.1% bovine serum albumin (BSA).

The total β-amyloid sandwich ELISA, quantitating both β-amyloid (aa1-40) and β-amyloid (aa 1-42) consists of two monoclonal antibodies(mAb) to β-amyloid. The capture antibody, 266 [P. Seubert, Nature (1992)359:325-327], is specific to amino acids 13-28 of β-amyloid. Theantibody 3D6 [Johnson-Wood et al., PNAS USA (1997) 94:1550-1555], whichis specific to amino acids 1-5 of β-amyloid, is biotinylated and servedas the reporter antibody in the assay. The 3D6 biotinylation procedureemploys the manufacturer's (Pierce, Rockford Ill.) protocol forNHS-biotin labeling of immunoglobulins except that 100 mM sodiumbicarbonate, pH 8.5 buffer is used. The 3D6 antibody does not recognizesecreted amyloid precursor protein (APP) or full-length APP but detectsonly β-amyloid species with an amino terminal aspartic acid. The assayhas a lower limit of sensitivity of ˜50 pg/ml (11 pM) and shows nocross-reactivity to the endogenous murine β-amyloid peptide atconcentrations up to 1 ng/ml.

The configuration of the sandwich ELISA quantitating the level ofβ-amyloid (aa 1-42) employs the mAb 21F12 [Johnson-Wood et al., PNAS USA(1997) 94:1550-1555] (which recognizes amino acids 33-42 of β-amyloid)as the capture antibody. Biotinylated 3D6 is also the reporter antibodyin this assay which has a lower limit of sensitivity of ˜125 pg/ml (28pM).

The 266 and 21F12 capture mAbs are coated at 10 pg/ml into 96 wellimmunoassay plates (Costar, Cambidge Mass.) overnight at roomtemperature. The plates are then aspirated and blocked with 0.25% humanserum albumin in PBS buffer for at least 1 hour at room temperature,then stored desiccated at 4° C. until use. The plates are rehydratedwith wash buffer (Tris-buffered saline, 0.05% Tween 20) prior to use.The samples and standards are added to the plates and incubatedovernight at 4° C. The plates are washed 3 times with wash bufferbetween each step of the assay. The biotinylated 3D6, diluted to 0.5μg/ml in casein incubation buffer (0.25% casein, PBS, 0.05% Tween 20, pH7.4) is incubated in the well for 1 hour at room temperature. Avidin-HRP(Vector, Burlingame Calif.) diluted 1:4000 in casein incubation bufferis added to the wells for 1 hour at room temperature. The colormetricsubstrate, Slow TMB-ELISA (Pierce, Cambridge Mass.), is added andallowed to react for 15 minutes, after which the enzymatic reaction isstopped with addition of 2 N H₂SO₄. Reaction product is quantified usinga Molecular Devices Vmax (Molecular Devices, Menlo Park Calif.)measuring the difference in absorbance at 450 nm and 650 nm.

2. Blood Assay

The EDTA plasma is diluted 1:1 in specimen diluent (0.2 gm/l sodiumphosphate.H₂O (monobasic), 2.16 gm/l sodium phosphate.7H₂O (dibasic),0.5 gm/l thimerosal, 8.5 gm/l sodium chloride, 0.5 ml Triton X-405, 6.0g/l globulin-free bovine serum albumin; and water). The samples andstandards in specimen diluent are assayed using the total β-amyloidassay (266 capture/3D6 reporter) described above for the brain assayexcept the specimen diluent was used instead of the casein diluentsdescribed.

From the foregoing description, various modifications and changes in thecomposition and method will occur to those skilled in the art. All suchmodifications coming within the scope of the appended claims areintended to be included therein.

1. The compound (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one.
 2. A pharmaceutical composition comprising (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one and a pharmaceutically acceptable diluent.
 3. A method of using (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting β-amyloid peptide release and/or its synthesis.
 4. A method of using (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating Alzheimer's disease.
 5. A method of using (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for preventing Alzheimer's disease.
 6. A method of using (N)-((S)-2-hydroxy-3-methyl-butyryl)-1-(L-alaninyl)-(S)-1-amino-3-methyl-4,5,6,7-tetrahydro-2H-3-benzazepin-2-one, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting the progression of Alzheimer's disease.
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. (canceled) 